Proteins, peptides and other drug molecules for therapeutic use are currently available in suitable forms in adequate quantities largely as a result of the advances in recombinant DNA technologies. The availability of such peptides and proteins has engendered advances in protein formulation and chemical modification. Chemical modification of biologically active peptides, proteins, oligonucleotides and other drugs for purposes of extending the serum half-life of such bioactive agents has been extensively studied. The ability to extend the serum half-life of such agents allows for the therapeutic potential of the agent to be realized without the need for high dosages and frequent administration.
Chemical modification used to extend the half-lives of proteins in vivo includes the chemical conjugation of a water soluble polymer, such as polyethylene glycol (PEG), to the protein of interest. A variety of approaches have been used to attach the polyethylene glycol molecules to the protein (PEGylation). For example, Royer (U.S. Pat. No. 4,002,531) states that reductive alkylation was used for attachment of polyethylene glycol molecules to an enzyme. Davis et al. (U.S. Pat. No. 4,179,337) disclose PEG:protein conjugates involving, for example, enzymes and insulin. Shaw (U.S. Pat. No. 4,904,584) disclose the modification of the number of lysine residues in proteins for the attachment of polyethylene glycol molecules via reactive amine groups. Hakimi et al. (U.S. Pat. No. 5,834,594) disclose substantially non-immunogenic water soluble PEG:protein conjugates, involving for example, the proteins IL-2, interferon alpha, and IL-1ra. The methods of Hakimi et al. involve the utilization of unique linkers to connect the various free amino groups in the protein to PEG. Kinstler et al. (U.S. Pat. Nos. 5,824,784 and 5,985,265) teach methods allowing for selectively N-terminally chemically modified proteins and analogs thereof, including G-CSF and consensus interferon.
Other approaches designed to extend the serum half-life of bioactive agents include: conjugation of the peptides to a large, stable protein which is too large to be filtered through the kidneys (e.g., serum albumin); G. D. Mao et al., Biomat., Art. Cells, Art. Org. 17:229-244 (1989); use of low- and high-density lipoproteins as transport vehicles and to increase serum half-life; P. Chris de Smidt et al., Nuc. Acids. Res., 19(17):4695-4700 (1991); the use of the Fc region of immunoglobulins to produce Fc-protein fusions; PCT WO 98/28427 (Mann et al, and references cited therein); and the use of the Fc domain to increase in vivo half-life of one or more biologically active peptides; PCT WO 00/24782 (Feige et al, and references cited therein).
Transthyretin (TTR) (formerly called prealbumin) is a 56 kDa tetrameric serum protein that plays important physiological roles as a transporter of thyroxine and retinol-binding protein; Hamilton and Benson, Cell. Mol. Life Sci., 58:1491-1521 (2001), and references cited therein. Blaney et al., in U.S. Pat. No. 5,714,142, describe the exploitation of TTR by endowing the drug to be administered with functionality that allows it to bind specifically to the protein. Specifically, Blaney et al. demonstrate that covalent attachment of a peptide, protein, nucleotide, oligonucleotide, oligosaccharide or other drug to a transthyretin-selective ligand will reversibly bind the drug to TTR and thereby increase the serum half-life of the agent based on the affinity of the ligand for TTR. It is stated that the intrinsic activity of the drug is not adversely affected and the resulting drug-TTR ligand conjugate will still be small enough to be orally absorbed.